人钾离子通道调节蛋白的原核表达及纯化

目的:探讨人钾离子通道调节蛋白(KCNRG)的原核表达体系和纯化条件,为该蛋白的功能机制研究提供工具。方法:以pcDNA3.1-KCNRG-2ex重组质粒为模板,采用特异引物扩增KCNRG基因编码区。PCR 产物双酶切后克隆入pET28a-MBP-His和pGEX-4T-1 载体中。重组的pET28a-KCNRG-MBP-His和pGEX-4T-1-KCNRG质粒转化Top10感受态细胞。鉴定阳性克隆,并选取测序正确的质粒转化Rosetta(DE3)蛋白表达菌株,IPTG 诱导表达,用SDS-PAGE 及Western blot检测重组融合蛋白的表达。结果: PCR扩增产物长度为819 bp。经双酶切和测序,鉴定重组质粒pET28a-KCNRG-MBP-His和pGEX-4T-1-KCNRG构建成功。SDS-PAGE检测,KCNRG-MBP融合蛋白在菌液的上清中表达,KCNRG-GST融合蛋白主要以包涵体形式表达。Western blot 检测,重组KCNRG-MBP蛋白和重组KCNRG-GST蛋白分别能被抗6×His 抗体和抗GST抗体识别。经镍柱亲和层析纯化,获得纯化的KCNRG-MBP融合蛋白(凝胶成像系统软件分析纯化蛋白的纯度>90%)。结论:成功构建重组质粒pET28a-KCNRG-MBP-His,并实现KCNRG-MBP融合蛋白的可溶性原核表达。

Prokaryotic expression and purification of human potassium channel regulator protein

Objective: To investigate the prokaryotic expression and purification conditions of human potassium channel regulator protein(KCNRG),providing tools for the study on its function and underlying mechanism. Methods: The coding region of KCNRG gene was amplified with specific primers from recombinant pcDNA3.1-KCNRG-2ex plasmid.The PCR products were cloned into the pET28a-MBP-His and pGEX-4T-1 vectors after double-enzyme digestion and ligation.The recombinant pET28a-KCNRG-MBP-His and pGEX-4T-1-KCNRG plasmids were transformed into Top10 cells.The positive clones were selected and sequenced.The correct clone of pET28a-KCNRG-MBP-His and pGEX-4T-1-KCNRG were transformed into prokaryotic expression strain Rosetta (DE3)and induced by IPTG.The expression of recombinant KCNRG fusion protein was analyzed by SDS-PAGE and Western blot. Results: The length of PCR product was 819 bp.The recombinant pET28a-KCNRG-MBP-His and pGEX-4T-1-KCNRG plasmids were confirmed by double restriction enzyme digestion and sequencing.The SDS-PAGE results showed that the KCNRG-MBP fusion protein was expressed in Rosetta(DE3)and soluble in supernatant and the KCNRG-GST fusion protein was mainly expressed in the form of inclusion bodies.The Western blot results revealed that the recombinant KCNRG-MBP protein and recombinant KCNRG-GST protein could be recognized by anti-6×His and anti-GST antibodies,respectively.The purified KCNRG-MBP fusion protein was obtained by nickel affinity chromatography with the purity >90% by gel imaging systerm software. Conclusion: The recombinant plasmid pET28a-KCNRG-MBP-His is successfully constructed,and soluble KCNRG-MBP fusion protein is obtained with the prokaryotic expression system.